杜氏盐藻电击转化方法的系统优化

An optimal electroporation system for Dunaliella salina

摘要:

本研究系统分析了盐藻生长状态、电击条件、电击缓冲液成分和质粒浓度等条件对电击转化效率的影响。实验结果表明: 正常接种后培养7 d对数生长中期的盐藻细胞, 在25 mF、0.8 kV的电击条件下加入终浓度为10 mg/mL的质粒可使盐藻电击转化效率达到1.85‰; 电击缓冲液中加入0.4 mol/L的甘油可使转化效率显著提高至2.03‰(P<0.05)。在上述优化电击体系下, 运用3种不同质粒分别转化盐藻细胞后获得的转化效率无显著差异。通过对电击转化中相关因素的优化, 本研究建立了一种适用于杜氏盐藻的高效稳定的电击转化体系, 为杜氏盐藻的转基因研究提供有效方法。

Abstract:

To optimize the electroporation system in Dunaliella salina (D. salina), we studied the effects of growth phase of cells, electric parameters, electroporation buffer and concentration of plasmid on transformation efficiency. The results showed that a transformation efficiency of 1.81‰ was achieved in D. salina cells at mid-log growth phase electroporated with plasmid (DCA-bar) 10 μg/mL, voltage 0.8 kV and capacitance 25 μF. However, when glycerol was added to electroporation buffer at a final concentration of 0.4 mol/L, the transformation efficiency was increased up to 2.03‰. Additionally, transformation was done with plasmids DCA-bar, NR-bar, pUΩ-bar respectively, under above optimum conditions, and similar transformation efficiencies were obtained. The findings indicate that an efficient and stable system of electroporation in D. salina has been developed, providing a powerful tool for the transgenic research of D. salina.