杜氏盐藻电击转化体系的优化

Optimization of an electroporation-mediated transform system of Dunaliella salina

[目的]构建盐藻高效遗传转化体系,以利于在细胞内组装靶标代谢途径,进而以盐藻为生物反应器规模化生产类胡萝卜素、生物燃油等高值产品。[方法]以杜氏盐藻(Dunaliella salina)为受体,以来源于高油植物斑鸠菊(Vernonia galamensis)编码DGAT酶的基因VgDGAT1a为靶基因,以pCAMBIA3301为表达载体,利用电击法进行遗传转化,优化转化条件和相关参数,并对转化体进行分子检测。[结果]杜氏盐藻对除草剂草铵膦敏感,固体和液体培养筛选阳性转化体的草铵膦浓度分别为20mg·L^(-1)和40mg·L^(-1)。优化的盐藻电转化技术条件包括:培养7d的藻细胞为受体,质粒浓度6mg·L^(-1),电击参数为0.4kV和4ms。盐藻转化率达2.63‰,比现有转化效率提高约1.14倍。分子检测显示靶基因VgDGAT1a基因成功转入杜氏盐藻并高效表达。[结论]建立的优化电击转化方法显著提高了盐藻转化效率,在高等植物遗传转化中,常用的抗除草剂草铵膦Bar基因可用作盐藻基因转化的选择标记,植物表达载体pCAMBIA3301亦可用于盐藻遗传转化。

[Objective]The present study was conducted to construct a highly-efficient genetic transformation system of Dunaliella salina,which would greatly facilitate the assembly of target metabolic pathways in cells,and further promote the large-scale production of high-valued products such as carotenoids and biofuels using Dunaliella salina as the bioreactor.[Methods]In this paper,Dunaliella salina was used as the target for genetic transformation.The foreign target gene was VgDGAT1 a which encode a DGAT protein,and a key enzyme responsible for TAG biosynthesis from high-oil plant Vernonia galamensis.The pCAMBIA3301 was selected as expression vector in microalgae.An electroporationmethod was selected for transformation and the related parameters was further optimized.Molecular testing tools were used to analyze the integration and expression of the target gene in transformants.[Result]Dunaliella salina was sensitive to herbicide glufosinate.Concentration of glufosinate was tested as 20 mg·L^（-1） and 40 mg·L^（-1） for effectively selecting the positive tranformant in solid and liquid culture media,respectively.The optimized electroporation conditions for Dunaliella salinatransformation included 7 dcultured microalgal cells as receptors,6 mg·L^（-1） plasmid per transformation,and setting of 0.4 kV and 4 ms for electroperation.The transformation rate of Dunaliella salina was 2.63‰,which was 1.14 times higher than previously reported value.Molecular identification showed that the target gene VgDGAT1 a was successfully transferred into Dunaliella salina and also highly expressed.[Conclusion]The optimized electroporationmethod developed here significantly increased the transformation rate of Dunaliella salina.The herbicide glufosinate resistant Bar gene and pCAMBIA3301 expression vector widely used in higher plant transformation also can be successfully used in Dunaliella salina genetic transformation.