Nuclear transformation of the diatom Phaeodactylum tricornutum using PCR-amplified DNA fragments by microparticle bombardment

Abstract

We have developed a method for marine diatom transformation by microparticle bombardment using polymerase chain reaction (PCR)-amplified DNA fragments. We constructed a circular vector (approximately 5000. bp) containing an fcpA promoter from Phaeodactylum tricornutum, antibiotic-resistance genes and terminator from Cylindrotheca fusiformis (a "gene cassette"). Then the various lengths of linear vectors (+ 0-+ 1000 linear vectors) were then PCR-amplified from the circular plasmid. The transformants of P. tricornutum transfected with the linear vectors were obtained in the triplicate experiments. Transformation efficiencies using PCR-amplified short linear vectors containing the gene cassette and additional DNA regions of 0, 50, and 500. bp at both ends of the gene cassette (+ 0-+ 500 linear vectors) did not significantly differ from one another or from the efficiency of the + 1000 linear vector. Transformation efficiencies using the linear vectors were lower than that using the circular vector, but were not significantly different. The ratios of the number of transformants containing the whole region of the gene cassette to those of transformants transfected using linear vectors of various lengths were determined. An extension (≧ 50 bp) of DNA fragments was effective for introducing the whole region of the gene cassette into the genomic DNA. In using various amounts of the + 50 linear vector (37.5-300 fmol/shot), we observed that transformation efficiencies using 37.5. fmol (52.2 ng)/shot of the linear vector were not significantly different from those obtained using 300. fmol of the linear vector. The 300. fmol quantity was set considering the quantity of the circular plasmid (1 μg = approx 300 fmol) and the 37.5 fmol quantity was set for quick and easy preparation of approximately 500 ng of the linear short vector needed for triplicate transformation experiments in one PCR tube containing 50 μl of PCR cocktail. Integrating the gene cassette of the short linear vectors as well as that of the full length of the linear vector (+ 1000 linear vector) into the chromosomal DNA was determined using Southern blot analysis. The short linear vectors tended to result in smaller numbers of insertions than those of the supercoiled plasmid. This simple and time-saving transformation method using microparticle bombardment with PCR-amplified DNA fragments permitted both functional analysis of diatom-specific genes and development of diatom strains useful for further biotechnological applications.