Protoplast Transformation of Mortierella isabellina with Hygromycin B Resistance Plasmid PD4

Abstract:

A strain Mortierella isabellinaM6-22-4,which was sensitive to hygromycin B, was selected by treating parental spores with N-methyl-N'-Nitro-N-nitrsoguanidine(MNNG). Protoplasts of the strain Mortierella isabellina M6-22-4 were transformed successfully to hygromycin B resistance using the PD4 plasmid, which contains the Escherichia coli hph gene under the control of Mortierella alpina his H4.1 promoter. The PD4 plasmid was introduced by PEG/CaCl₂ treatment. Transformation frequencies of 1.6～2.8 transformants/μg of DNA were achieved. Then they were successively incubated to non-selected PDA plates for 10 generations. About 31.6% transformants only from digested plasmid were mitotically stable and showed different hygromycin B resistance when they were incubated back to selection plates. The results of PCR and Southern analysis in three transformants indicated that the plasmid PD4 had been integrated into the fungal genome with 1～2 copies. This is the first report of Mortierella isabellina transformation system and supplies an important tool for further research into genetic manipulation of this filamentous fungus.