以潮霉素抗性为选择标记的毛壳霉原生质体转化

Transformation of Chaetomium globosum with hygromycin B resistance plasmids pCB1003

利用 PEG方法 ,将含有潮霉素抗性标记的质粒 p CB10 0 3转化毛壳霉原生质体 ,并在高于毛壳霉敏感的潮霉素浓度下筛选转化子。转化率达 1～ 2个转化子 / μg质粒 DNA。以潮霉素抗性的毛壳霉转化子的基因组 DNA为模板进行 PCR扩增及其产物的 Southern杂交。结果表明 ,潮霉素抗性基因已整合入毛壳霉染色体。稳定性试验表明 。

Genetic transformation has been achieved by incubating protoplasts from Chaetomium globosum with plasmid PCB1003. The efficiency was 1 2 transformants/μg vector DNA. Protoplasts were produced by treating 14 hour old mycelium of Chaetomium globosum with 10 mg/mL lysozyme. Chaetomium globosum growth was inhibited completely when the medium contained 150 μg/mL hygromycin B. Integration of hph gene was verified by PCR and PCR product hybridization analysis. There was no evidence of mitotic of hygromycin B resistance in the transformants.