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.

中文翻译：

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基于农杆菌介导的转化方法的高效 RNAi 系统的开发，用于研究药用真菌 Wolfiporia cocos 的功能基因组学

报道的 Wolfiporia cocos 的遗传转化方法是有限的。在这项研究中，我们首次描述了一种基于农杆菌介导的 W. cocos 转化方法的高效 RNA 干扰 (RNAi) 系统。使用内源性乳清蛋白-5'-磷酸脱羧酶基因 (URA3) 作为选择标记和沉默基因，在来自香菇的 Legpd 和 Leactin 双启动子和单分别来自 W. cocos 的 Wcgpd 启动子。结果表明，两种启动子均有效驱动了URA3基因的表达，在含有5'-氟乳清酸的CYM培养基上可以筛选出URA3沉默的转化体。此外，沉默URA3基因对W. cocos菌丝的生长没有影响。在该研究中还观察到 URA3 基因座的不完全沉默。本研究将促进对椰枣中药理化合物的底物降解、菌核形成和生物合成网络机制的进一步研究。