AIM:To establish a convenient and efficient transformation system of the marine-sourced filamentous fungus Phoma herbarum YS4108.METHODS:The endogenous actin promoter was cloned with degenerate primers and genome walking strategy,and utilized in the electroporation of P.herbarum YS4108.Using hygromycin B phosphotransferase gene as a selection marker and single cells prepared from mycelia as recipients,the effect of electroporation parameters on transformation efficiency was examined.RESULTS:The actin promoter was successfully cloned.The optimum parameters for electroporation were found to be 0.75 Kv (0.2 cm cuvette) in voltage and 400 Ω in resistance with 9 μg of plasmid,under which more than 100 transformants per cuvette (106 recipients) could be obtained.CONCLUSION:By using the endogenous actin promoter,the electric transformation system of P.herbarum YS4108 was established,which served as a practical approach for transgenic study on this fungus.%目的:建立一种简便有效的海洋丝状真菌Phoma herbarum YS4108的转基因方法.方法:用简并引物扩增和染色体步行克隆P.herbarum YS4108的actin启动子,以潮霉素抗性基因为选择标记,用部分原生质体化的单细胞进行电转化并摸索最佳电转化参数.结果:成功获得了actin启动子序列.并确定了最佳电转化参数:电压0.75 kV(0.2 cm 电转化杯),电阻400Ω,载体DNA用量为9 μg.在此条件下可获得多于100个转化子/电转化杯(106细胞).结论:内源性的actin启动子可用于P.herbarum YS4108的电转化,该方法可用于P.herbarum的转基因研究.
展开▼
