中国生物工程杂志

Phaeodactylum tricornutum is a potential source of biodiesel for its abundance in fatty acids. Moreover as its fatty acid composition contains much eicosapentaenoic acid (EPA, 20:5D5,8,11,14,17), P. tricornutum represents an interesting alternative source for the industrial production. The absence of an adapted transformation system has been a serious limitation for its genetic manipulation. A novel transformation method was firstly established using micro-particle bombardment in P. tricornutum. The results showed that the GUS gene, a reporter gene, was successfully expressed. Transgenetic cells presented blue color under the microscope after stained. In addition, different factors which influenced transformation were optimized. The experiment indicated that the best parameters for P. tricornutu transformation: 60 µg plasmid DNA was coated onto the tungsten particles, 1500psi rupture-disc pressures was applied at a bombardment distances of 6 cm.. This newly method overcame the obstacle of P. tricornutum rigid cell walls. The adoption of endogenetic fcp promoter facilitated the expression of foreign gene. The P. tricornutum was tested for sensitivity to 5 antibiotics: kananycin( Km), ampicillin (Amp), streptomycin (Str), neomycin(Nm) and chloramphenicol(Cm). The result of sensitivity test showed that P. tricornutum were not sensitive to Km, Amp, Str and Nm. Otherwise, P. tricornutum is highly sensitive to Cm, the half lethal concentration of Cm in liquid culture was 60μg/ml. It suggests that Cm can be suitable to select regent for P. tricornutum genetic engineering. This method can be used as a potential tool in the research of P. tricornutu gene engineering and modification of its fatty acids synthesis pathway.