利用电激共转化法转化橡胶树悬浮细胞的初步研究

利用电激转化法对橡胶树悬浮细胞系进行转化,初步确定卡那霉素的筛选浓度、共转化的实验条件。通过电激共转化法将NPTII、GUS、EGFP和Hb CBF1基因成功导入到橡胶树悬浮细胞并整合到基因组中。通过卡那霉素抗性筛选、GFP荧光观察和PCR验证等方法,确定外源DNA片段转化成功。共筛选得到95个抗性愈伤组织,其中9个愈伤组织经过PCR验证确定整合有外源DNA片段,1个愈伤组织共转化有NPTII和GUS基因。此结果表明电激共转化法可成功应用于橡胶树悬浮细胞转化操作。

A Preliminary Study of Co-transforming Rubber Tree Suspension Cells using Electroporation

The rubber tree suspension cells were co-transformed using electroporation method. The co-transforming conditions and kanamycin concentration were determined. The NPTII, GUS, eGFP, and HbCBF1 genes were successfully transferred into suspension cells and integrated into genomes in rubber tree. The transgenes were validated by kanamycin resistance selection, GFP fluorescence detection and PCR amplification methods. Totally 95 kanamycin resistant calli were obtained, in which 9 harbored exogenous DNA fragments basing on the PCR validaton results. One callus was co-transformed with NPTII and GUS genes. These results demonstrated that electroporation co-transform method could be successfully applied in genetic manipulation in rubber tree.