An Efficient PEG/CaCl2-Mediated Transformation Approach for the Medicinal Fungus Wolfiporia cocos

Sun, Qiao; Wei, Wei; Zhao, Juan; Song, Jia; Peng, Fang; Zhang, Shaopeng; Zheng, Yanyan; Chen, Ping; Zhu, Wenjun

doi:10.4014/jmb.1501.01053

Cited by 15 publications

(6 citation statements)

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“…In Ganoderma lucidum , another traditional Chinese medicinal mushroom, over-expression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene by using Agrobacterium tumefaciens -mediated transformation method led to a two fold increase in ganoderic acid content, increased accumulation of intermediates and the up-regulation of downstream genes ( Xu et al, 2012 ), indicating that genetic engineering is an efficient approach to manipulate the economic traits of fungi. Furthermore, an efficient and stable genetic transformation system for W. cocos has been developed ( Sun et al, 2015 ), and the genes and pathways involved in triterpenoids (the main active compound in W. cocos ) biosynthesis are known ( Shu et al, 2013 ). Based on our analysis, the orthologs of TPK2, HOG1, FUS3, SLT2, STE11, and STE7 in other fungi play significant roles in sclerotial development, and they are upregulated during the sclerotial stage ( Figure 2 ; Supplementary Table S1 ), indicating that these PKs potentially positively regulate sclerotial formation in W. cocos .…”

Section: Discussionmentioning

confidence: 99%

The Kinome of Edible and Medicinal Fungus Wolfiporia cocos

et al. 2016

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Wolfiporia cocos is an edible and medicinal fungus that grows in association with pine trees, and its dried sclerotium, known as Fuling in China, has been used as a traditional medicine in East Asian countries for centuries. Nearly 10% of the traditional Chinese medicinal preparations contain W. cocos. Currently, the commercial production of Fuling is limited because of the lack of pine-based substrate and paucity of knowledge about the sclerotial development of the fungus. Since protein kinase (PKs) play significant roles in the regulation of growth, development, reproduction, and environmental responses in filamentous fungi, the kinome of W. cocos was analyzed by identifying the PKs genes, studying transcript profiles and assigning PKs to orthologous groups. Of the 10 putative PKs, 11 encode atypical PKs, and 13, 10, 2, 22, and 11 could encoded PKs from the AGC, CAMK, CK, CMGC, STE, and TLK Groups, respectively. The level of transcripts from PK genes associated with sclerotia formation in the mycelium and sclerotium stages were analyzed by qRT-PCR. Based on the functions of the orthologs in Sclerotinia sclerotiorum (a sclerotia-formation fungus) and Saccharomyces cerevisiae, the potential roles of these W. cocos PKs were assigned. To the best of our knowledge, our study is the first identification and functional discussion of the kinome in the edible and medicinal fungus W. cocos. Our study systematically suggests potential roles of W. cocos PKs and provide comprehensive and novel insights into W. cocos sclerotial development and other economically important traits. Additionally, based on our result, genetic engineering can be employed for over expression or interference of some significant PKs genes to promote sclerotial growth and the accumulation of active compounds.

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Section: Discussionmentioning

confidence: 99%

The Kinome of Edible and Medicinal Fungus Wolfiporia cocos

et al. 2016

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“…The PEG-mediated protoplast transformation was modified as previously described ( Sun et al, 2015 ). Each 100 μL of protoplasts was mixed with constructed sgRNA-Cas9 plasmid and placed on ice for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Genome editing in the edible fungus Poria cocos using CRISPR-Cas9 system integrating genome-wide off-target prediction and detection

et al. 2022

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Poria cocos is an important edible and medicinal fungus with a long history. However, the lack of adequate genetic tools has hindered molecular genetic research and the genetic modification of this species. In this study, the endogenous U6 promoters were identified by mining data from the P. cocos genome, and the promoter sequence was used to construct a sgRNA expression vector pFC332-PcU6. Then, the protoplast isolation protocol was developed, and the sgRNA-Cas9 vector was successfully transformed into the cells of P. cocos via PEG/CaCl2-mediated transformation approach. Off-target sites were genome-widely predicted and detected. As a result, the target marker gene ura3 was successfully disrupted by the CRISPR-Cas9 system. This is the first report of genome editing in P. cocos using CRISPR-Cas9 system integrating genome-wide off-target prediction and detection. These data will open up new avenues for the investigation of genetic breeding and commercial production of edible and medicinal fungus.

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“…The PEG-mediated protoplast transformation was performed as previously described with minor modi cations [43,44]. Protoplasts were digested with 1.5% lysozyme (Guangdong Culture Collection Center, Guangzhou, China) at 80 rpm for 2 h at 28°C and ltered through repeated washing with 0.6 M mannitol.…”

Section: Peg-mediated Protoplast Transformationmentioning

confidence: 99%

Functional validation of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing

Liu

Xie

et al. 2023

Preprint

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Background Squalene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids. In recent years, CRISPR/Cas9 gene editing technology has emerged and other functional genes in the Poria cocos triterpene synthesis pathway have been studied; PcSE has not been reported. Results In this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. sgRNA sequences were designed based on the active site residues. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. Which showed that deletion of PcSE inhibited triterpene synthesis. Conclusions A gene editing system based on molecular docking was successfully constructed to demonstrate that PcSE functions as a house squalene cyclooxygenase, which provides a basis for further studies on the heterologous biosynthesis of P. cocos secondary metabolites.

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An Efficient PEG/CaCl2-Mediated Transformation Approach for the Medicinal Fungus Wolfiporia cocos

Cited by 15 publications

References 0 publications

The Kinome of Edible and Medicinal Fungus Wolfiporia cocos

The Kinome of Edible and Medicinal Fungus Wolfiporia cocos

Genome editing in the edible fungus Poria cocos using CRISPR-Cas9 system integrating genome-wide off-target prediction and detection

Functional validation of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing

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