Construction of an RNAi expression vector and transformation into Penicillium chrysogenum

摘要

In this work, we constructed an RNAi vector for attenuation of the class III chitin synthase gene chs4, which plays a major role in hyphal growth and conidia formation. To achieve a high transformation frequency, factors affecting the preparation and regeneration of protoplasts were analyzed. The maximum numbers of protoplasts (1.41?×?107?mL?1) were released when mycelia cultured for 48?h were incubated at 30?°C for 5?h in a buffer containing 4?mg?mL?1 lysing enzyme. The maximum regeneration rate (33?%) was obtained when mycelia were digested for 4?h and plated on a regeneration medium containing 1?% overlaid agar. Quantitative real-time PCR was performed to validate the transformation efficiency, and it revealed knockdown of chs4 gene in randomly selected transformants at different levels. Dramatic reductions in the formation of conidia and the hyphal growth rate were observed in most of the transformants.