除草剂草丁膦抗性基因bar作为三角褐指藻基因工程选择标记的研究

研究了三角褐指藻(Phaeodactylum tricornutum)对氯霉素、卡那霉素、青霉素、链霉素、潮霉素5种抗生素和一种除草剂草丁膦的敏感性,发现三角褐指藻对草丁膦非常敏感,用统计学分析法得出了其72h半致死质量浓度为22mg/L。通过基因枪法将携带草丁膦抗性基因bar的载体pSVB导入三角褐指藻细胞中,通过草丁膦筛选成功获得了抗性藻细胞,PCR检测获得阳性结果,提示草丁膦更适合用于构建安全、高效、廉价的三角褐指藻表达系统。

Herbicide basta resistant gen bar as a new selective marker for genetic engineering of Phaeodactylum tricornutum

Zeocin, a routine selective reagent in diatom genetic engineering, is not suitable for the construction of expression system of diatoms, due to its photosensitivity and mutagenicity. In order to find a suitable selective reagent for photobioreactor-based culture of transformed Phaeodactylum tricornutum. The sensitivity of P. tricornutum to five antibiotics (Chloramphenicol, Kanamycin, Penicillin, Streptomycin, and Hygromycin) and phosphinothricin (PPT) was tested. The results showed that P. tricornutum was sensitive to PPT, with a 72-h half death concentration of 22 mg/L. The expression vector pSVB, which contained PPT-resistant gene bar, was transformed into P. tricornutum cells by particle bombardment; and the resistant P. tricornutum was selected by PPT screening. It was perimentally confirmed that the transformed P. tricornutum was resistent to PPT. This research indicates that phosphinothrin is suitable for constructing a safe, efficient, low cost expression system of P. tricornutum.