Optimization of protoplast preparation and transformation conditions of antagonistic Trichoderma strain gz-2

ObjectiveIn this study,protoplast preparation and transformation conditions of antagonistic Trichoder-ma strain gz-2 were optimized to obtain strains marked by green fluorescent tag rapidly and effectively,provide reference for GFP tags of T. harzianum and other fungi of the same kind,and lay a foundation for the study of colonization dynamics, distribution regulation and biocontrol features.MethodThe conditions of protoplast preparation and transformation of T. harzianum strain gz-2 was optimized with the protoplast transformation system mediated by PEG-CaCl2. The condi-tions were optimized from following aspects: age and digestion time of T. harzianum strain,content of plasmid in the transformation system and the concentration of hygromycin B(HmB) in regeneration medium. Transformants whose expressions were stable were obtained ,and the growth features of them were compared with the original strains to evaluate transfon effects.ResultProtoplast prepared by hypha of strain gz-2 cultured for 36 h was the most stable. The number of protoplast(11.67 × 106 protoplast/mL) was the largest when mycelium enzymolysis time was 3.5 h. When plasmid DNA content was 40μL in every 240μL transformation system,the obtained transformant number was the larg-est,reaching 8.67 transformant/plate. When HmB content in regeneration medium plate was 600μg/mL,stable strong transformants could be obtained.ConclusionA stable transformant GFP-gz-2 strain is obtained which has sound genetic stability. There is no significant difference in colony morphology,hypha growth rate and spore quantity between it and original strain gz-2.