Institute of Applied Mycology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
Key Laboratory of Agro-Microbial Resource Comprehensive Utilization, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, China.
World J Microbiol Biotechnol. 2020 Aug 16;36(9):140. doi: 10.1007/s11274-020-02916-0.
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.
报道的药用真菌槐栓菌遗传转化方法有限。本研究首次描述了一种基于农杆菌介导转化法的槐栓菌有效 RNA 干扰（RNAi）系统。利用内源性尿嘧啶 5'-磷酸脱羧酶基因（URA3）作为选择标记和沉默基因，在香菇 Legpd 和 Leactin 双启动子以及槐栓菌 Wcgpd 单启动子的控制下，将生长活跃的菌丝块作为转化受体。结果表明，两种启动子均可有效驱动 URA3 基因的表达，并且可以在含有 5'-氟乳清酸的 CYM 培养基上选择 URA3 沉默转化体。此外，沉默 URA3 基因对槐栓菌菌丝的生长没有影响。本研究还观察到 URA3 基因座的不完全沉默。该研究将促进进一步研究槐栓菌在底物降解、菌核形成和药理化合物生物合成网络方面的机制。
