The development of an efficient RNAi system based on Agrobacterium -mediated transformation approach for studying functi
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(2020) 36:140
ORIGINAL PAPER
The development of an efficient RNAi system based on Agrobacteriummediated transformation approach for studying functional genomics in medical fungus Wolfiporia cocos Naiyao Chen1 · Mengting Chen1 · Ting Wu1 · Yinbing Bian2 · Zhangyi Xu1,2 Received: 3 April 2020 / Accepted: 11 August 2020 © Springer Nature B.V. 2020
Abstract 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. Keywords Wolfiporia cocos · gene introduction · RNA interfere · URA3 · gene function
Introduction Wolfiporia cocos is a well-known sclerotium-forming fungus that was widely used for its medicinal and culinary value. The dried sclerotium of W. cocos, named fuling in Chinese and bukuryo in Japanese, have been documented to be used as a crude sedative, stomachic, and diuretic in traditional Chinese and Japanese herbal medicine for thousands of years, and it has been popularly added to nutraceuticals, tea supplements, wine supplements, cosmetics, and functional Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11274-020-02916-0) contains supplementary material, which is available to authorized users. * Zhangyi Xu [email protected] 1
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
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foods in the world (Wang et al. 2012). In previous years, several reports have demonstrated that the polysaccharides and triterpenoids of W. cocos function as two major categories of pharmacological compounds with multiple immunostimulatory, antitumor, anti-inflammatory, and invigorating activities (Ukiya et al. 2002; Wang et al. 2012; Shi et al. 2017; Lee et al. 2017). Although W. cocos sclerotium have been commercially cultivated in China for several hundred years (Wang et al. 2013), there are still the problems of instability o
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