The development of an efficient RNAi system based on Agrobacterium-mediated transformation approach for studying...

Genetic transformation methods reported for Wolfiporia cocos are limited. In this study, we describe an efficient RNA interference (RNAi) system based on Agrobacterium-mediated transformation approach in W. cocos for the first time. Actively growing mycelial plugs were used as recipients for transformation using endogenous orotidine-5'-phosphate decarboxylase gene (URA3) as both a selective marker and a silencing gene, under the control of the dual promoters of Legpd and Leactin from Lentinula edodes and the single promoter of Wcgpd from W. cocos, respectively. The results showed that both the two kinds of promoters effectively drive the expression of URA3 gene, and the URA3-silenced transformants could be selected on CYM medium containing 5'-fluoroorotic acid. In addition, silencing URA3 gene has no effect on the growth of W. cocos hyphae. The incomplete silencing of the URA3 locus was also observed in this study. This study will promote further study on the mechanism of substrate degradation, sclerotial formation, and biosynthesis network of pharmacological compounds in W. cocos.