The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.

Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5'-monophosphate decarboxylase gene (URA3) was cloned as a silencing...

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Autores principales:	Dashuai Mu, Liang Shi, Ang Ren, Mengjiao Li, Fengli Wu, Ailiang Jiang, Mingwen Zhao
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/14e5d6672a4646639e3f86611e8efb99
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spelling	oai:doaj.org-article:14e5d6672a4646639e3f86611e8efb992021-11-18T07:07:42ZThe development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.1932-620310.1371/journal.pone.0043737https://doaj.org/article/14e5d6672a4646639e3f86611e8efb992012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22937087/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5'-monophosphate decarboxylase gene (URA3) was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%). To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes.Dashuai MuLiang ShiAng RenMengjiao LiFengli WuAiliang JiangMingwen ZhaoPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 8, p e43737 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Dashuai Mu Liang Shi Ang Ren Mengjiao Li Fengli Wu Ailiang Jiang Mingwen Zhao The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.
description	Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5'-monophosphate decarboxylase gene (URA3) was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%). To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes.
format	article
author	Dashuai Mu Liang Shi Ang Ren Mengjiao Li Fengli Wu Ailiang Jiang Mingwen Zhao
author_facet	Dashuai Mu Liang Shi Ang Ren Mengjiao Li Fengli Wu Ailiang Jiang Mingwen Zhao
author_sort	Dashuai Mu
title	The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.
title_short	The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.
title_full	The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.
title_fullStr	The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.
title_full_unstemmed	The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.
title_sort	development and application of a multiple gene co-silencing system using endogenous ura3 as a reporter gene in ganoderma lucidum.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/14e5d6672a4646639e3f86611e8efb99
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The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.

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