三角褐指藻基因枪转化体系的建立

Establishment of a Micro-particle Bombardment Transformation System for Phaeodactylum tricornutum

三角褐指藻具有较高的脂肪酸含量,是一种很有潜力的生物柴油生产原料。此外,它是多不饱和脂肪酸尤其是二十碳五烯酸(EPA)重要的来源。合适转化体系的缺乏限制了通过基因工程手段对其进行改造。首次采用基因枪方法成功地将外源基因转入三角褐指藻,转化细胞经染色后呈现蓝色,表明外源报告基因β-糖苷酸酶(GUS)基因得到了成功的表达。同时还进行了转化参数等因素对转化效率影响的分析,优化了转化条件。结果显示最佳的转化条件为:每60μg钨粉包被1μg质粒DNA,样品室真空度为27英寸汞柱,可裂膜为1500psi,受体与阻挡网距离6cm。此外,转化载体采用了三角褐指藻内源基因fcp的启动子,实现了外源基因在细胞内的表达。通过5种基因工程中常用抗生素对三角褐指藻生长抑制的研究发现,三角褐指藻对卡那霉素、氨苄青霉素、链霉素和新霉素不敏感,500mg/L的卡那霉素、氨苄青霉素和新霉素,以及1000mg/L链霉素仍不能抑制其生长;三角褐指藻对氯霉素非常敏感,130mg/L的氯霉素可以完全抑制其生长,其半抑制浓度为60mg/L。这为基因工程手段改造三角褐指藻脂肪酸代谢相关途径奠定了基础。

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 ceils 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 6cm. This new 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 euhure 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.