小球藻和莱茵衣藻原生质体的电转化研究

A Study on Electro-transformation in Protoplasts of Chlorella and Chlamydomonas

以小球藻及莱茵衣藻原生质体为受体细胞,利用电击法将质粒p CAMBIA1301转入小球藻和莱茵衣藻,摸索电击转化条件并进行分子检测。结果表明:两类藻都对潮霉素敏感,小球藻及莱茵衣藻分别在含25 mg/L和100 mg/L潮霉素的固体培养基上的生长被完全抑制;小球藻和莱茵衣藻原生质体电击转化的最佳电击场强分别为0.8 k V/cm和0.6 k V/cm,最佳脉冲时间均为10 ms;制备原生质体和通过2-脱氧-D-葡萄糖处理可明显提高转化效率;分子检测说明GUS报告基因成功转入两种藻并可稳定遗传。

The plasmid pCAMBIA1301 was transformed into The receptor cells Chlorella vulgaris and Chlamydomonas reinhardtii protoplasts by electroporation method.The related parameters was tested and molecular detections were per-formed in transformants.The results showed that：two kinds of algae were sensitive to hygromycin.The growth of Chlo-rella and Chlamydomonas was completely inhibited on solid media containing 25 mg/L and 100 mg/L concentration of hygromycin respectively.The optimal electric field intensity were 0.8 kV/cm and 0.6 kV/cm in Chlorella and Chlamydomonas respectively.The optimal pulse time was 10 ms for both species.Making protoplasts and treating with 2-deoxy-D-glucose make it possible to significantly increase the transformation rates.Molecular identification indicated that GUS gene has been successfully transformed into the two species of algae and the genetic form is stable.