盐生杜氏藻RNAi应用中的电激转化条件

Electroporation Transformation Condition of RNAi Application in Dunaliella salina

采用了两种电激模式-高电场强度低电容模式下3～7kV／cm和低电场强度高电容模式下300-500V／cm转化质粒pBl221-cat；PCR扩增检测cat基因部分序列及组织化学染色法GUS检测也同时证明质粒pBl221-cat在盐生杜氏藻中瞬时表达。在此基础之上，插入pds基因的正反向重复序列，构建RNAi表达载体pBIRNAI-Dsa，电激方法转入杜氏盐藻细胞，荧光定量PCR结果表明，转入干涉载体pBIRNAI-Dsa的实验组的pds基因表达显著下降，最低达对照组的28％，表明表达受到抑制。

Plasmid pBI211-cat was transferred into D. salina via electroporation and was proved transiently expressed. Vector pBIRNAI-Dsa was constructed from pBI211-cat and transformed into Dunalilla salina inducing pds gene silencing. Two modes, high-voltage and low-pitched of 3-7 kV/cm and low-volt- age and high-tension of 300-500 V/cm, were adopted to transform ceils and the results were detected from different aspects. PCR amplification and GUS histochemistry staining also proved that pBI221-cat had been delivered into cells and expressed transiently. Partial pds sense sequence and its reverse were inserted into pBI221-cat to form the RNAi vector pBIRNAI-Dsa. Pds gene expressing of the treated group transformed with pBIRNAI-Dsa was reduced, even to 28% of the control group, suggesting that pds gene expressing was restrained.