Abstract:

Objective This study aims to optimize the protoplast-mediated transformation system of Penicillium marneffei,it would provide a good platform for the gene function research of P.marneffei.Methods We inserted the selectable marker pyrG gene (from A.nidulans) into P.marneffei strain ligD (pyrG-,ligD-) by protoplast method,screened positive transformant by medium without uracil,and used PCR to verify the restructuring.Through changing some important effecting factors including enzymolysis period,concentration of plasmid,protoplast fusion agents PTC and screening culture medium to find out the optimal protocol.Results As a result,The positive transformants growed well in the media without uracil,and PCR analysis showed that pyrG was inserted into the transformants,the appropriate conditions for protoplast-mediated transformation of P.marneffei were as follows:enzymatic time is 6 h;PTC:60%PEG-4000,100 mmol/L Tris-HCl pH8.0,100 mmol/L CaCl₂;0.6 mol/L sucrose screening culture medium;add 2.5 μg plasmid per 100 μL protoplast (10⁶~10⁷/mL).The transformation efficiency was about 68 transformants/μg plasmid DNA under these conditions.Conclusions Protoplasts-mediated transformation in P.marneffei was successfully optimized,this method could get high transformation efficiency and provide a good platform for P.marneffei gene function research.

Key words: Penicillium marneffei, protoplast, transformation