Enhanced frequency of Beauveria bassiana blastospore transformation by restriction enzyme-mediated integration and electroporation.
The techniques of restriction enzyme-mediated integration (REMI) and electroporation (EP) were applied for the first time to improving the blastospore transformation of fungal biocontrol agent Beauveria bassiana for higher frequency. The blastospores from < or =24 h incubation in glucose-mineral medium after shaking conidia for 48 h in Subouraud dextrose broth were found most competent for integrating 1 microg plasmid DNA vectoring the phosphinothricin (PPT) resistance gene bar in 360 microL reaction system containing 100 U HindIII or XbaI. Such blastospores were also most suitable for EP transformation at the optimized field strength of 10 kV cm(-1). The optimized REMI and EP generated averagely 39 and 53 transformants microg(-1) plasmid DNA whereas polyethylene glycol (PEG) integration yielded only 22. All transformants grew well on Czapek's agar containing 400 microg PPT mL(-1) after three rounds of cultivation on the same agar excluding PPT but their parental strain showed no resistance. The target gene inserted into the genomes of 10 transformants randomly taken from REMI or EP transformation was consistently detected by both PCR and Southern blotting. Compared to the PEG integration, REMI and EP enhanced the frequency of the blastospore transformation by 73 and 137%, respectively.
Jiang
Qiong
Q
Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, PR China.
Ying
Sheng-Hua
SH
Feng
Ming-Guang
MG
D000613:Aminobutyrates / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D052982:Beauveria / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D015247:Deoxyribonuclease HindIII / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D025141:Drug Resistance, Fungal / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D005656:Fungal Proteins / Q000235:genetics; D018014:Gene Transfer Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D013172:Spores, Fungal / Q000235:genetics*; D014170:Transformation, Genetic
D000613:Aminobutyrates; D000935:Antifungal Agents; D005656:Fungal Proteins; C003121:phosphinothricin; D015247:Deoxyribonuclease HindIII; C025580:endodeoxyribonuclease XBAI; D015252:Deoxyribonucleases, Type II Site-Specific